Engineered proteins, such as bi- or multispecific antibodies capable of binding two or more antigens are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
A wide variety of recombinant multispecific antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g. an IgG antibody format and single chain domains (see e.g. Coloma, M. J., et. al., Nature Biotech. 15 (1997) 159-163; WO 2001/077342; and Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234).
In one approach, bispecific antibodies that are very similar to natural antibodies have been produced using the quadroma technology (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different antibody species are generated of which only one is the desired, functional bispecific antibody. Due to the presence of mispaired byproducts, and significantly reduced production yields, means for sophisticated purification procedures are required (see e.g. Morrison, S. L., Nature Biotech. 25 (2007) 1233-1234). In general the same problem of mispaired byproducts remains if recombinant expression techniques are used.
In WO 98/50431 common light chains are used in multispecific antibodies to prevent mispairing of light and heavy chains, however the approach according to WO 98/50431 uses different heavy chains which are heterodimerized via the so-called ‘knobs-into-holes’ technology (Ridgway, J. B., Protein Eng. 9 (1996) 617-621; and WO 96/027011). In WO 98/50431 high yields of antibodies with heterodimerized (‘knob-hole’) heavy chains was observed. However, some homodimer formation (‘hole-hole’ or ‘knob-knob’) was also observed. The percentage of heterodimerized heavy chains could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and introducing a disulfide bridge between both CH3 domains in order to stabilize the heterodimers (Merchant A. M, et al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). New approaches using the principle of the knobs-into-holes technology are described e.g. in EP 1870459A1. One important constraint of this strategy is that the light chains of the two parent antibodies have to be 100% identical to prevent mispairing and formation of inactive molecules. The development of common light chains fitting to denovo generated antibodies is still challenging. Thus, this technique is not appropriate for easily developing recombinant, bispecific antibodies against two antigens starting from two different antibodies against the first and the second antigen, as either the heavy chains of these antibodies and/or the identical light chains have to be optimized.
WO 2012/023053 relates to bispecific antibodies using a common heavy chain. This approach is even more restricted instead of being generally applicable given the difficulties for generating common chains in general, and specifically as the binding properties for the bispecific antibodies have to be conferred only via the two different light chains directed against the first and second antigen without any contribution of the heavy chain. This is s clear constraint in view of the fact that in the majority of antibodies the heavy chain hypervariable regions, especially e.g. the complementarity determining region 3 of the heavy chain (CDR3-H), are attributed to be important for the binding properties of antibodies to their target antigen.
WO 2009/080252 relates to bivalent, bispecific antibodies, wherein in only one of the two antibody arms, the heavy chain variable domain (VH) and the light chain variable domain (VL) are exchanged in order to prevent light chain mispairing by generating light chains built up of different domains.